The ends of mammalian chromosomes terminate in long arrays of TTAGGG repeats that have associated with them specific DNA binding proteins. These nucleoprotein complexes are known as telomeres and they function to preserve the integrity of chromosomes during the cell cycle by allowing the proper segregation during cell division. More specifically, telomeres shield the chromosome ends from degradation or end-on fusion, prevent the activation of DNA damage checkpoints, and modulate activity of telomerase, an enzyme that maintains the length of telomeres. Without this enzymatic activity, telomeres shorten at each cell division because the replication machinery fails to replicate DNA ends.
Human somatic cell chromosomes undergo normal telomere shortening at each cell division, and it is believed that this process is a tumor suppression mechanism that limits the number of potential replication cycles of the cell. However, in some cancers it has been demonstrated that telomeres do not undergo replication-associated shortening resulting in the transformed phenotype and the subsequent development of tumorigenesis. On the other hand, the complete loss of telomeric DNA in tumors has also been observed, collectively suggesting that any modification to telomere length homeostasis can contribute to the carcinogenic phenotype. For this reason, much effort is currently focused on the characterization of factors responsible for maintaining the normal status of telomeres within the cell (Muniyappa and Kironmai, Crit. Rev. Biochem . Mol. Biol., 1998, 33, 297-336).
Telomeric Repeat Binding Factor 1 (also known as TRF1 and TRBF1) is a protein component of the telomere nucleoprotein complex that binds to double-stranded telomeric repeat arrays and anchors them to the nuclear matrix (Luderus et al., J. Cell. Biol., 1996, 135, 867-881; Zhong et al., Mol. Cell. Biol., 1992, 12, 4834-4843). Telomeric Repeat Binding Factor 1 colocalizes with telomeric DNA in interphase and metaphase cells and it is believed that Telomeric Repeat Binding Factor 1 suppresses telomere elongation by physically interfering with the association of telomerase at the chromosome ends (Chong et al., Science, 1995, 270, 1663-1667; van Steensel and de Lange, Nature, 1997, 385, 740-743). Further support for a steric mechanism of action includes studies demonstrating that Telomeric Repeat Binding Factor 1 can form dimers and tetramers, and that when these multimers bind DNA they induce conformational changes in the telomeres (Bianchi et al., Embo. J., 1997, 16, 1785-1794). In addition, it has also been shown that Telomeric Repeat Binding Factor 1 multimers induce the formation of parallel telomeric tracts along the sites of Telomeric Repeat Binding Factor 1 binding (Griffith et al., J. Mol. Biol., 1998, 278, 79-88).
Two forms of Telomeric Repeat Binding Factor 1, generated by alternative splicing and differing by 60 base pairs, have been isolated and characterized (Shen et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 13618-13623; Young et al., Somat. Cell. Mol. Genet., 1997, 23, 275-286). PIN2, the alternative splice variant of Telomeric Repeat Binding Factor 1, lacks amino acids 296-316 found in Telomeric Repeat Binding Factor 1 and was shown to be the predominant species in HeLa cells. In addition PIN2 was shown to be cell cycle regulated suggesting a role for PIN2 in mitosis (Shen et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 13618-13623).
Recently, it was shown that Telomeric Repeat Binding Factor 1 interacts with other proteins as well as DNA. Nosaka et al. identified human nucleoside diphosphate kinase nm23-H2 as a Telomeric Repeat Binding Factor 1 interacting protein using a yeast two-hybrid screen (Nosaka et al., Biochem. Biophys. Res. Commun., 1998, 243, 342-348). nm23-H2 was previously identified as a c-myc transcription factor that binds single-stranded DNA sequences that contain pyrimidine rich stretches.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Telomeric Repeat Binding Factor 1. To date, strategies aimed at modulating Telomeric Repeat Binding Factor 1 function have involved the use of antibodies, antisense molecules, and truncated forms of the protein that represent binding domains.
Disclosed in PCT application WO 97/08314 is the nucleotide sequence of Telomeric Repeat Binding Factor 1 and the protein encoded thereby. Also disclosed are expression vectors for the production of the protein and host cells that are transfected with these vectors, antibodies to Telomeric Repeat Binding Factor 1 and ribozymes that cleave Telomeric Repeat Binding Factor 1. Also disclosed are antisense nucleic acid molecules, both DNA and RNA in nature, that hybridize to the Telomeric Repeat Binding Factor 1 mRNA initiation codon or any portion of a Telomeric Repeat Binding Factor 1 mRNA (De Lange, 1997).
The modular nature of the Telomeric Repeat Binding Factor 1 protein, containing several distinct functional domains, has led to the investigation of altered or truncated forms of the protein as possible inhibitors. Disclosed in PCT application WO 99/15662 are the nucleic acid sequence of Telomeric Repeat Binding Factor 1 and the isolated nucleic acid sequences that comprise and code for the various domains within Telomeric Repeat Binding Factor 1. Also disclosed is the protein and proteolytic fragments thereof (De Lange et al., 1999). Antisense oligonucleotides and ribozymes are generally disclosed. Disclosed in U.S. Pat. No. 5,733,730 are the TRF protein, methods to isolate the protein and active truncated fragments of the protein that constitute defined DNA binding domains (De Lange, 1998). Truncated forms of the nucleic acid encoding Telomeric Repeat Binding Factor 1 are also disclosed in PCT application WO 98/36066 and U.S. Pat. No. 5,859,183 as are vectors and host cells that express the truncated variants and methods to identify the heterodimers formed between the truncated forms and the native Telomeric Repeat Binding Factor 1 protein (De Lange et al., 1999; De Lange et al., 1998).
There remains a long felt need for agents capable of effectively inhibiting Telomeric Repeat Binding Factor 1 function and antisense technology is emerging as an effective means for reducing the expression of specific gene products. Therefore, this technology may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Telomeric Repeat Binding Factor 1 expression.
The present invention provides compositions and methods for modulating Telomeric Repeat Binding Factor 1 expression, including modulation of the truncated form of Telomeric Repeat Binding Factor 1, also known as PIN2.